Azaanthraquinone compound with tobacco mosaic virus resistance and its preparation method and application

Abstract

This invention discloses a nitrogen-containing heterocyclic anthraquinone compound with anti-tobacco mosaic virus activity, its preparation method, and its applications. The molecular formula of the compound is: C 22 H 19 NO4, the compound is named: 5-methyl-3,6-dimethoxy-2-(3'-methyl-1'-) H 3,6-dimethoxy-5-methyl-2-(3'-methyl-1'-yl)anthracene-9,10-dione, also known as pyrrole-2'-yl)anthracene-9,10-dione. H The compound of this invention, denoted as -pyrrol-2'-yl)anthracene-9,10-dione, has the following structure: This compound was first isolated from the Dai medicinal herb *Cassia tora*. This nitrogen-containing heterocyclic anthraquinone compound exhibits excellent anti-tobacco mosaic virus (Tobacco Mosaic Virus) activity: in experiments against Tobacco Mosaic Virus, its relative inhibition rate reached 56.2%, significantly higher than the relative inhibition rate of the control compound ningnanmycin (32.4%). The compound of this invention has a stable structure and good activity, showing promising application prospects in the preparation of anti-Tobacco Mosaic Virus drugs and can serve as a lead compound for anti-Tobacco Mosaic Virus drugs.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of natural product chemistry, and particularly relates to a nitrogen heterocyclic anthraquinone compound with anti-tobacco mosaic virus activity extracted from Cassia L. plants of Cassia bihacida, and application thereof in preparation of anti-tobacco mosaic virus drugs. BACKGROUND

[0002] Plant viruses are the second largest pathogenic organisms in nature after fungi. It is very difficult to find a drug that can inhibit viruses without damaging the host, so the prevention and treatment of plant viral diseases is very difficult. Almost every crop and economic crop is infected by viral diseases, which has caused serious losses to agricultural production. Taking tobacco mosaic virus (TMV) as an example, TMV has the characteristics of wide host range, strong stress resistance and great production hazards, and can infect more than 260 plants in 38 families. After the tobacco plant is infected by tobacco mosaic virus, the color of the tobacco leaf after baking is uneven, the tobacco aroma and taste are poor, and the quality is greatly reduced. The high incidence of mosaic disease has caused great losses to agriculture and seriously affected the development of agriculture and the income of farmers.

[0003] In recent years, the research on the antiviral activity of natural plants at home and abroad has increased, and it plays an important role in the prevention and treatment of human, animal and crop diseases. It can also be expected that in the future for a long period of time, the development of efficient and safe viral inhibitors from natural active substances such as plant sources, microbial sources and animal sources will still be the focus of natural product research. There are more than 600 species of Cassia L. plants in the world, which are widely distributed in the tropical and subtropical regions of the earth, especially in tropical America and tropical Africa. There are 25 species in China, widely distributed in southern provinces and regions, and all species are distributed in Yunnan. Many Cassia L. plants can be used as medicine, of which five species of Cassia L. plants (Cassia fistula, Cassia bihacida, Cassia siamea, Cassia siamensis and Cassia obtusifolia) are commonly used as Dai medicine by the Dai people in Yunnan. Traditional folk medicine shows that these plants have the effects of clearing heat and detoxifying, promoting water and removing stones, relieving swelling and pain, relieving wind and pain, killing insects and relieving itching, etc. The representative chemical components of Cassia L. plants are chromones and nitrogen heterocyclic anthraquinones; these compounds show various activities such as anti-cancer, anti-HIV virus, anti-hepatitis, insecticidal, anti-anxiety and sedative.

[0004] Anthraquinones are quinone compounds. Anthraquinone derivatives are widely found in natural products and can also be synthesized artificially. Anthraquinones include their products and dimers at different degrees of reduction, such as anthraquinones, oxyanthraquinones, and anthrones, as well as the glycosides of these compounds. In natural products, anthraquinones are commonly found in the metabolites of higher and lower plants, lichens, and fungi, and have hemostatic, antibacterial, detoxifying, laxative, and diuretic effects. This invention discovers a novel nitrogen-containing heterocyclic anthraquinone compound from the legume *Cassia tora*, a species of the genus *Cassia*. Notably, this compound is a rare one containing 3'-methyl-1'... H A novel anthraquinone skeletal molecule with a pyrrole structural fragment, exhibiting high structural novelty and significant anti-tobacco mosaic virus activity. Summary of the Invention

[0005] The first objective of this invention is to provide a nitrogen-containing heterocyclic anthraquinone compound with anti-tobacco mosaic virus activity; the second objective is to provide a method for preparing the aforementioned nitrogen-containing heterocyclic anthraquinone compound with anti-tobacco mosaic virus activity; and the third objective is to provide applications of the aforementioned nitrogen-containing heterocyclic anthraquinone compound with anti-tobacco mosaic virus activity.

[0006] The first objective of this invention is achieved as follows: the nitrogen-containing heterocyclic anthraquinone compound with anti-tobacco mosaic virus activity is prepared from *Cassia tora* as raw material through pretreatment, extract extraction, MCI decolorization, silica gel column chromatography, and high-performance liquid chromatography separation steps. The molecular formula of the nitrogen-containing heterocyclic anthraquinone compound with anti-tobacco mosaic virus activity is: C 22 H 19 NO4, named as: 5-methyl-3,6-dimethoxy-2-(3'-methyl-1'-) H 3,6-dimethoxy-5-methyl-2-(3'-methyl-1'-yl)anthracene-9,10-dione, also known as pyrrole-2'-yl)anthracene-9,10-dione. H -pyrrol-2'-yl)anthracene-9,10-dione has the following structure:

[0007] .

[0008] The second objective of this invention is achieved by using the fine branches of the Dai medicinal herb *Cassia tora* as raw material, and preparing it through pretreatment, extract extraction, MCI decolorization, silica gel column chromatography, and high-performance liquid chromatography separation steps, specifically including:

[0009] A. Pre-treatment: Crush the fine branches of Cassia biloba and pass them through a 20-60 mesh sieve to obtain material a;

[0010] B, extract infusion: the material a is added with 2-6 times of organic extraction solvent of the material a, soaked and extracted at normal temperature for 2-5 times, and each time is 12-20 hours, the extract liquid is combined and filtered, and then the filtrate is concentrated and added with the same volume of ethyl acetate to extract 2-4 times, and then concentrated to obtain sample extract b;

[0011] C, MCI decolorization: the sample extract b is concentrated and decolorized on an MCI column, the effluent is collected and concentrated under reduced pressure to obtain extract c;

[0012] D, silica gel column chromatography:

[0013] 1) 3-10 times of 200-300 mesh silica gel is added to the extract c, and the gradient elution is carried out with chloroform-methanol solution with a volume ratio of 20:1-1:1, and the same parts are combined by TLC monitoring;

[0014] 2) the eluate obtained by eluting with 8:2 chloroform-methanol solution is further separated by silica gel column chromatography with 2.5-10 times of 200-300 mesh silica gel, and then gradient elution is carried out with chloroform-acetone solution, and the eluate is collected, and the same parts are combined by TLC monitoring;

[0015] E, gel column chromatography purification: the 3:2 chloroform-acetone eluate is concentrated, then dissolved with methanol, and then purified by Sephadex LH-20 dextran gel column chromatography to obtain the target compound of the nitrogen heterocyclic anthraquinone compound with anti-tobacco mosaic virus activity;

[0016] F, high performance liquid chromatography separation: the target compound of the nitrogen heterocyclic anthraquinone compound with anti-tobacco mosaic virus activity is separated and purified by high pressure liquid chromatography to obtain the target compound of the nitrogen heterocyclic anthraquinone compound with anti-tobacco mosaic virus activity.

[0017] The structure of the nitrogen heterocyclic anthraquinone compound with anti-tobacco mosaic virus activity prepared in the above steps can be identified by the following method:

[0018] Appearance observation: the compound of the present application is a light red gel; ultraviolet-visible absorption spectrum shows that it has maximum absorption at 212, 260, 284, 408 nm, which proves that the compound contains aromatic ring structure; infrared spectrum (potassium bromide tablet) shows that the compound contains -NH (3358 cm -1 , 3126 cm -1 ), ketone carbonyl (1665 cm -1 ), aromatic ring (1618, 1524, 1439 cm -1) Characteristic functional groups; High resolution mass spectrometry (HRESIMS) gave a quasi-molecular ion peak at 384.1217 [M+Na] + , the molecular formula of the compound was determined as C 22 H 19 NO4.

[0019] Table 1. Characteristic data of the compounds 1 HNMR and 13 CNMR data (CDC13)

[0020]

[0021] According to the1H NMR spectrum, the compound contains six aromatic protons δ H 7.97 (s, H-1), 7.37 (s, H-4), 6.95 (d, 7.8 Hz, H-7), 7.76 (d, 7.8 Hz, H-8), 6.19 (d, 2.8 Hz, H-4') and 6.73 (d, 2.8 Hz, H-5'), two methyl protons δ δ H 1.89 (s, H3-6) and 2.13 (s, H3-7'), two methoxy protons δ H 3.80 (s, 3-OCH3) and 3.84 (s, 6-OCH3), and one amino proton δ H 7.61 (s, NH). 13 C NMR, DEPT and HSQC spectra showed that there were 22 carbon atoms, including 2 carbonyl, 10 aromatic non-proton substituted carbon atoms, 6 aromatic quaternary carbon atoms, 2 methoxy carbon atoms and 2 methyl carbon atoms. The HMBC correlation of H-1 with C-4a / C-3 and C-9, H-4 with C-la / C-2 and C-10, H-7 with C-5 and C-9a, H-8 with C-10a / C-6 and C-9 Figure 3 ) indicated the presence of a 2,3,5,6-tetrasubstituted 9,10-anthraquinone skeleton. Further HMBC correlations from H-4' with C-2', H-5' with C-2' and C-3', H3-6' with C-2' / C-3' and C-4', and H-NH with C-3' and C-4', together with the remaining three degrees of unsaturation proved the presence of a 3-methyl-1H-pyrrole. The 3-methyl-1 H -pyrrole was located at C-2 by the correlation of H-NH and C-2 and H-1 and C-2. Figure 3) remaining substitution positions were determined by HMBC correlations of H3-3-OCH3 with C-3, H3-6-OCH3 with C-6 and H3-7' with C-5. Thus, the structure of the compound was determined as 5-methyl-3,6-dimethoxy-2-(3'-methyl-1'-pyrrol-2'-yl)anthracene-9,10-dione. H - pyrrol-2'-yl)anthracene-9,10-dione. H - pyrrol-2'-yl)anthracene-9,10-dione.

[0022] The infrared, ultraviolet and mass spectral data of the compound: UV (methanol) lmax (log e) 212 (4.46), 260 (3.61), 284 (3.58), 408 (3.42) nm; IR (KBr) n max 3358, 3126, 3057, 2938, 2842,1665, 1618, 1524, 1439, 1342, 1135, 1064, 769 cm -1 ; 1 H and 13 C NMR data are shown in Table 1; HRESIMS (positive mode) m / z 384.1217 [M+Na] + ( calculated value C 22 H 19 NNaO4, 384.1212).

[0023] Through literature search, the compound of the present application is first discovered in natural products, and is an anthraquinone molecule with a 3'-methyl-1'-pyrrol structure fragment, which has high novelty in structure. H - pyrrol-2'-yl)anthracene-9,10-dione.

[0024] The third object of the present application is achieved by the use of the azacyclic anthraquinone compound in the preparation of a medicine for resisting tobacco mosaic virus.

[0025] The third object of the present application, the evaluation of the tobacco mosaic virus resisting activity of the compound, is achieved as follows:

[0026] The tobacco mosaic virus resisting activity test is carried out by the half-leaf method, and the tobacco mosaic virus resisting activity of the compound of the present application is determined at a mass concentration of 20 μM of the medicine. On 5-6 tobacco plants, the leaf suitable for the test (normal leaf row, no disease and no insect) is selected, the leaf is uniformly sprinkled with fine emery, and the prepared tobacco mosaic virus source (3.0 x 10 -3) Evenly spread on the sandblasted leaves, after all the selected leaves are treated with the poison, immediately put in the culture dish containing the liquid medicine for 20 min, take out, sprinkle the water droplets and about liquid on the leaves, put two half leaves in the enamel tray covered with wet paper, cover with glass, control the temperature (23±2) ℃, and place in the greenhouse under natural light, and the dry spots can be seen in 2-3 days. Another half leaf is set as a control for each treatment, and another group of treatments of the commercial ningnanmycin are set as a comparison, and the relative inhibition rate is calculated according to the following formula.

[0027] XI% = (CK-T) / CK * 100%

[0028] X: relative inhibition rate (%), CK: the number of dry spots of the half leaf soaked in water (pieces), T: the number of dry spots of the half leaf soaked in the liquid (pieces).

[0029] The relative inhibition rate of the compound is 56.2%, which is higher than the relative inhibition rate 32.4% of the control ningnanmycin, indicating that the compound has good activity against tobacco mosaic virus.

[0030] The advantages of the present application are:

[0031] 1. The present application first separates a new compound from the Dai medicine Cassia siamea Lam, determines that the compound is an indole alkaloid compound by nuclear magnetic resonance and mass spectrometry determination method, and characterizes the specific structure, and the compound is a new azacycloanthraquinone compound with a 3'-methyl-1'- H pyrrole structure fragment first found in natural products. The compound has good activity against tobacco mosaic virus. Through the experiment of resisting tobacco mosaic virus, it is found that the relative inhibition rate of the indole alkaloid compound reaches 56.2%, and the activity is higher than that of the control ningnanmycin (32.4%). The above results reveal that the compound of the present application has good application prospect in preparing anti-tobacco mosaic virus drugs. The compound of the present application has simple structure and good activity, and can be used as a lead compound of anti-tobacco mosaic virus drugs.

[0032] 2. The compound of the present application is easy to obtain, the extraction method of the compound is simple, easy to separate, and easy to realize industrial production.

[0033] 3. The compound of the present application is easy to realize artificial synthesis, and the subsequent industrialization can also be realized through artificial synthesis.

[0034] 4. The preparation method of the compound of the present application adopts the combination of conventional column chromatography and high performance liquid chromatography, the operation process of the compound preparation is simple, the purity of the compound obtained is high, and the quality and purity of the compound in the subsequent industrial production are guaranteed.

[0035] 5. The compounds of this invention are safe and non-toxic, exhibiting good anti-tobacco mosaic virus activity, and can provide an ideal new type of drug molecule with a novel skeleton for the prevention and control of tobacco mosaic disease. Attached Figure Description

[0036] Figure 1 The carbon NMR spectra of the nitrogen-containing heterocyclic anthraquinone compounds of this invention ( 13 (C NMR).

[0037] Figure 2 The proton nuclear magnetic resonance spectra of the nitrogen-containing heterocyclic anthraquinone compounds of this invention ( 1 H NMR);

[0038] Figure 3 The key HMBC correlation diagram of the nitrogen-containing heterocyclic anthraquinone compounds of this invention. Detailed Implementation

[0039] The present invention will be further described below with reference to embodiments and accompanying drawings, but this does not limit the present invention in any way. Any modifications or substitutions made based on the teachings of the present invention shall fall within the protection scope of the present invention.

[0040] The nitrogen-containing heterocyclic anthraquinone compounds with anti-tobacco mosaic virus activity described in this invention are prepared from *Cassia tora* using *Cassia biloba* as a raw material through pretreatment, extract extraction, MCI decolorization, silica gel column chromatography, and high-performance liquid chromatography separation steps. The molecular formula of the nitrogen-containing heterocyclic anthraquinone compounds with anti-tobacco mosaic virus activity is: C 22 H 19 NO4, named as: 5-methyl-3,6-dimethoxy-2-(3'-methyl-1'-) H 3,6-dimethoxy-5-methyl-2-(3'-methyl-1'-yl)anthracene-9,10-dione, also known as pyrrole-2'-yl)anthracene-9,10-dione. H -pyrrol-2'-yl)anthracene-9,10-dione has the following structure:

[0041] .

[0042] The method for preparing the nitrogen-containing anthraquinone compound with anti-tobacco mosaic virus activity according to the present invention uses the fine branches of the Dai medicinal herb *Cassia tora* as raw material, and is prepared through pretreatment, extract extraction, MCI decolorization, silica gel column chromatography, and high-performance liquid chromatography separation steps, specifically including:

[0043] A. Pre-treatment: Crush the fine branches of Cassia biloba and pass them through a 20-60 mesh sieve to obtain material a;

[0044] B, extract infusion: adding 2-6 times of organic extraction solvent to the material a, soaking and extracting for 2-5 times at room temperature, each time for 12-20 hours, combining the extract and filtering, adding equal volume of ethyl acetate to extract 2-4 times after concentration, and concentrating to obtain sample extract b;

[0045] C, MCI decolorization: concentrating the sample extract b on the MCI column for decolorization, collecting the effluent and concentrating under reduced pressure to obtain extract c;

[0046] D, silica gel column chromatography:

[0047] 1) adding 3-10 times of 200-300 mesh silica gel to the extract c, gradient eluting with chloroform-methanol solution with a volume ratio of 20:1-1:1, and combining the same parts by TLC monitoring;

[0048] 2) adding 8:2 chloroform-methanol solution to the eluent to obtain 2.5-10 times of 200-300 mesh silica gel, further separating by silica gel column chromatography, and gradient eluting with chloroform-acetone solution, collecting each part of the eluent, and combining the same parts by TLC monitoring;

[0049] E, gel column chromatography purification: concentrating the 3:2 chloroform-acetone eluent, then dissolving with methanol, and purifying by Sephadex LH-20 dextran gel column chromatography to obtain the target product, crude azacyclononanoid compound with anti-tobacco mosaic virus activity;

[0050] F, high performance liquid chromatography separation: purifying the target product, azacyclononanoid compound with anti-tobacco mosaic virus activity, by high pressure liquid chromatography to obtain the target product, azacyclononanoid compound with anti-tobacco mosaic virus activity.

[0051] The organic extraction solvent in step B is 70-100% methanol aqueous solution, 70-100% ethanol aqueous solution or 65-100% acetone aqueous solution.

[0052] Before the silica gel column chromatography in step D, the extract c is diluted with 1.5-3 times of acetone or methanol, and then mixed with 1.5-2.5 times of 80-100 mesh silica gel.

[0053] In step D1), the volume ratio of chloroform to methanol in the chloroform-methanol solution is 20:1, 9:1, 8:2, 7:3, 6:4 and 5:5.

[0054] The volume ratio of chloroform and acetone in the chloroform-acetone solution in step 2) is 4:1, 3:2, 2:3, 1:4 and 1:9.

[0055] F In the step of high pressure liquid chromatography separation and purification, a 21.2 mm x 250 mm, 5 μ m Zorbax Prep HT GFC 18 chromatographic column is used, the flow rate is 12 mL / min, the mobile phase is a 56 wt% methanol-water solution, the ultraviolet detector detection wavelength is 408 nm, the sample injection amount is 0.5-2.0 m L, and the eluent corresponding to the chromatographic peak retention time of 30.8 min after each sample injection is collected and accumulated multiple times. After removing the solvent from the eluent, the nitrogen heterocyclic anthraquinone compound of the present application is obtained.

[0056] The application of the nitrogen heterocyclic anthraquinone compound of the present application is in the preparation of a tobacco mosaic virus resistant drug.

[0057] The specific operation of the preparation method is as follows:

[0058] A, sample extraction and purification: dry the twigs of Cassia siamea, crush them to 20-60 mesh, then extract them 2-5 times with a solvent, the solvent is 2-6 times the weight of the raw material, the extraction time is 12-20 hours, filter out the precipitate to obtain a sample extract, and concentrate under reduced pressure to obtain a extract. The prepared ethanol extract is partitioned in ethyl acetate and an aqueous solution. The ethyl acetate layer is concentrated under reduced pressure to obtain a crude extract. The obtained crude extract is filtered to remove the precipitate, and then subjected to decolorization on an MCI column. The effluent is collected and concentrated under reduced pressure to obtain an extract for gel purification. The part enriched with the nitrogen heterocyclic anthraquinone compound obtained by gel purification is prepared for chromatographic separation.

[0059] B, first silica gel column chromatography: the obtained extract is diluted with 1.5-3 times the amount of acetone or methanol, then mixed with silica gel in an amount of 1.5-2.5 times the weight of the extract, and the mixed silica gel is 80-120 mesh. The mixed sample is subjected to column chromatography on silica gel, the silica gel used for column chromatography is 200-300 mesh, and the weight of the silica gel is 3-10 times the weight of the extract; gradient elution is performed with a mixed organic solvent of chloroform and methanol in a volume ratio of 20:1-1:1, the gradient eluate of each gradient is collected and concentrated, and TLC monitoring is performed to combine the same parts to obtain 5 components (A-E).

[0060] C, second silica gel column chromatography: the component b (chloroform-methanol 8:2 elution part) in step B is further subjected to silica gel column chromatography: the silica gel used for column chromatography is 200-300 mesh, and the weight of the silica gel is 2-10 times the weight of the extract; gradient elution is performed with a mixed organic solvent of chloroform and acetone in a volume ratio of 4:1-1:9, and the gradient eluate of each gradient is collected and concentrated.

[0061] D, gel purification, the component b (eluted by chloroform-acetone 3:2 mixed organic solvent) in step C is subjected to gel purification, and column chromatography is carried out by using Sephadex LH-20 dextran gel as the stationary phase, and methanol as the mobile phase.

[0062] E, high performance liquid chromatography separation: the enriched part of the azacyclononquinone compound in the present application obtained in step D is subjected to separation and purification by high performance liquid chromatography, and the azacyclononquinone compound in the present application is obtained.

[0063] Further, preferably, the solvent in step A is acetone aqueous solution with a volume concentration of 70-100%, ethanol aqueous solution with a volume concentration of 70-100%, or methanol aqueous solution with a volume concentration of 70-100%.

[0064] Further, preferably, before the extract in step C is subjected to column chromatography by using silica gel, the extract is diluted by using acetone or methanol with a weight of 1.5-3 times of the extract, and then the sample is mixed with 80-100 mesh silica gel with a weight of 1.5-2.5 times of the extract, and then the sample is loaded.

[0065] Further, preferably, in step C, when gradient elution is carried out, the volume ratio of chloroform and methanol mixed organic solvent used is 20:1, 9:1, 8:2, 7:3, 6:4 and 5:5 in turn.

[0066] Further, preferably, in step E, the high performance liquid chromatography separation and purification is carried out by using methanol aqueous solution with a volume concentration of 50-65% as the mobile phase, a flow rate of 12 mL / min, a Zorbax PrepHT GF reversed-phase preparation column with a size of 2.12*250 mm and a particle size of 5 μm as the stationary phase, a UV detector with a detection wavelength of 376 nm, a sample injection of 0.5-1.0 mL each time, and a collection of chromatographic peaks in 30-42 min, and the compound pure product is obtained after multiple accumulations and evaporation.

[0067] The raw material of Dai medicine Cassia ambigua used in the present application is not limited by regions and varieties, and can be used to realize the present application,

[0068] The raw material of Dai medicine Cassia ambigua from different origins in Yunnan is used to further illustrate the present application as follows.

[0069] Example 1

[0070] The preparation method of the anti-tobacco mosaic virus active azacyclononquinone compound in the present application comprises the steps of extract preparation, silica gel column chromatography and high performance liquid chromatography separation, and the Dai medicine Cassia ambigua twig is used as the raw material, and the specific operation is as follows:

[0071] The Dai medicine Cassia torosa used is produced in Honghe Estuary, Yunnan. The raw material is the fine branches of Cassia torosa. 6.4 kg of the sample after being crushed to 30 meshes is soaked and extracted with 70% acetone aqueous solution for 4 times, each time for 15 h. The extract is combined and then extracted with an equal volume of ethyl acetate for 3 times. The extract is combined and then decolorized by MCI column. The eluent is concentrated under reduced pressure to obtain extract. The extract is dissolved in 2 times the mass of methanol, and then mixed with 90 mesh silica gel. After mixing, the sample is loaded on a column. The sample is eluted with chloroform-methanol eluent with a volume ratio of 20:1, 8:2, 7:3, 6:4 and 5:5, respectively. The gradient eluent is collected, concentrated, and monitored by TLC. The same parts are combined to obtain 5 parts A-E. Among them, the collected sample B (8:2) part 285 g is loaded on a column with 8 times the mass of 200-300 mesh silica gel. The sample is eluted with chloroform-acetone eluent with a volume ratio of 4:1, 3:2, 2:3, 1:4 and 1:9, respectively. The gradient eluent is collected, concentrated, and monitored by TLC. The same parts are combined to obtain 6 parts. Among them, the 3:2 part 42.5 g is further separated by column chromatography with methanol as the mobile phase. The part enriched with azacyclanthenylquinone compounds obtained by gel column chromatography is further separated by column chromatography with 56% methanol as the mobile phase, a flow rate of 12 ml / min, a column size of 2.12*250 mm, and a column temperature of 25-30°C. The eluent is collected, concentrated, and dried to obtain the anti-tobacco mosaic virus active anthraquinone compound. µ m Zorbax PrepHT GF reversed-phase preparative column as the stationary phase, and a UV detector with a detection wavelength of 408 nm. Each time, 0.8 mL is injected, and the chromatographic peak of 30.8 min is collected. After multiple accumulations, it is dried to obtain the anti-tobacco mosaic virus active anthraquinone compound.

[0072] The structure of the prepared anti-tobacco mosaic virus active azacyclanthenylquinone compound is identified by the following method:

[0073] Appearance observation shows that the compound of the present application is a light red gel; UV-visible absorption spectrum shows that it has maximum absorption at 212, 260, 284 and 408 nm, proving that there is an aromatic ring structure in the compound; infrared spectrum (potassium bromide tablet) shows that the compound has -NH (3358 cm -1 , 3126 cm -1 ), ketone carbonyl (1665 cm -1 ), and aromatic ring (1618, 1524, 1439 cm -1 ) characteristic functional groups; high-resolution mass spectrometry (HRESIMS) gives a quasi-molecular ion peak of 384.1217 [M+Na] + , which can determine the molecular formula of the compound as C22 H 19 NO4。

[0074] According to the1H NMR spectrum, the compound contains six aromatic protons δ H 7.97 (s, H-1), 7.37 (s, H-4), 6.95 (d, 7.8 Hz, H-7), 7.76 (d, 7.8 Hz, H-8), 6.19 (d, 2.8 Hz, H-4') and 6.73 (d, 2.8 Hz, H-5'), two methyl δ H 1.89 (s, H3-6) and 2.13 (s, H3-7'), two methoxy protons δ H 3.80 (s, 3-OCH3) and 3.84 (s, 6-OCH3), and one amino proton δ H 7.61 (s, NH). 13 The1H NMR, DEPT and HSQC spectra showed a total of 22 carbon atoms, including 2 carbonyl, 10 aromatic non-proton substituted carbon atoms, 6 aromatic quaternary carbon atoms, 2 methoxy carbon atoms and 2 methyl carbon atoms. The HMBC correlations of H-1 with C-4a / C-3 and C-9, H-4 with C-la / C-2 and C-10, H-7 with C-5 and C-9a, H-8 with C-10a / C-6 and C-9 ( Figure 3 ) indicated the presence of a 2,3,5,6-tetrasubstituted 9,10-anthraquinone skeleton. Further HMBC correlations from H-4' with C-2', H-5' with C-2' and C-3', H3-6' with C-2' / C-3' and C-4', and H-NH with C-3' and C-4', together with the remaining three degrees of unsaturation proved the presence of a 3-methyl-l H -pyrrole. The location of this 3-methyl-l H -pyrrole on C-2 was determined by the correlation of H-NH and C-2 and H-1 and C-2. Figure 3 ) The remaining substitution positions were determined by the HMBC correlations of H3-3-OCH3 with C-3, H3-6-OCH3 with C-6 and H3-7' with C-5. Thus, the structure of the compound was determined to be 5-methyl-3,6-dimethoxy-2-(3'-methyl-l' H -pyrrole-2'-yl)anthracene-9,10-dione, with the English name 3,6-dimethoxy-5-methyl- 2-(3'-methyl-l' Hpyrrol-2'-yl)anthracene-9,10-dione.

[0075] Example 2

[0076] The present example provides a preparation method of the tobacco mosaic virus active nitrogen heterocyclic anthraquinone compound according to the present application, which comprises the steps of extractive extraction, silica gel column chromatography and high performance liquid chromatography separation, and uses medicinal plant Cassia siamea as raw material, and the specific operation is as follows:

[0077] The medicinal plant Cassia siamea used is from Yuanjiang, Yuxi, Yunnan, and the raw material is Cassia siamea branchlet, which is crushed to 30 meshes, and 8.6 kg of the sample is soaked and extracted with 75% acetone aqueous solution for 3 times, 12 h each time. The extractive is combined and then extracted with equal volume of ethyl acetate for 3 times. The extractive is then decolorized by MCI column, and the eluent is concentrated under reduced pressure to obtain extractive; the extractive is dissolved with 2 times the mass of methanol, and then 80-120 mesh silica gel is added for mixing, and the mixture is loaded on a column packed with 200-300 mesh silica gel; gradient elution is performed with chloroform-methanol eluent with volume ratio of 20:1, 8:2, 7:3, 6:4 and 5:5, respectively, the gradient eluent is collected, concentrated, and monitored by TLC, and the same parts are combined to obtain 5 parts A-E; among them, the collected sample B (8:2) part 342 g is loaded on a column packed with 250 mesh silica gel with 6 times the mass of the extractive, and gradient elution is performed with chloroform-acetone eluent with volume ratio of 4:1, 3:2, 2:3, 1:4 and 1:9, respectively; the gradient eluent is collected, concentrated, and monitored by TLC, and the same parts are combined to obtain 5 parts, and among them, the 3:2 part 56.3 g is further separated by column chromatography with methanol as mobile phase. The part enriched with nitrogen heterocyclic anthraquinone compound obtained by gel column chromatography separation is further separated by column chromatography with 56% methanol as mobile phase, at a flow rate of 12 ml / min, 2.12x250 mm, 5 µ m Zorbax PrepHT GF reversed-phase preparation column as stationary phase, and an ultraviolet detector with detection wavelength of 408 nm; 1.5 mL of sample is loaded each time, and the chromatographic peak at 30.8 min is collected; after multiple accumulations, it is evaporated to dryness to obtain the tobacco mosaic virus active anthraquinone compound according to the present application.

[0078] Example 3

[0079] The present example provides a preparation method of the tobacco mosaic virus active nitrogen heterocyclic anthraquinone compound according to the present application, which comprises the steps of extractive extraction, silica gel column chromatography and high performance liquid chromatography separation, and uses medicinal plant Cassia siamea as raw material, and the specific operation is as follows:

[0080] The medicinal plant Cassia siamea was produced in Menghai, Xishuangbanna, Yunnan. The raw material was the fine branches of Cassia siamea. After being crushed to 50 mesh, 9.4 kg of the sample was soaked in 70% aqueous acetone solution for extraction for 4 times, each for 15 hours. The extract was combined and then extracted with ethyl acetate for 3 times. The extract was combined and then decolorized by MCI column. The eluent was concentrated under reduced pressure to obtain extract. The extract was dissolved in methanol with the amount of methanol being 2 times the weight of the extract. Then, the sample was mixed with silica gel of 80-120 mesh. After mixing, the sample was loaded on a column filled with silica gel of 200-300 mesh. The sample was eluted with chloroform-methanol eluent with the volume ratio being 20:1, 8:2, 7:3, 6:4 and 5:5, respectively. The gradient eluent was collected and concentrated. The same parts were combined after TLC monitoring. Six parts A-F were obtained. Among them, the sample B (8:2) part 462 g was collected. The sample was loaded on a column filled with 200-300 mesh silica gel with the amount of silica gel being 7 times the weight of the extract. The sample was eluted with chloroform-acetone eluent with the volume ratio being 4:1, 3:2, 2:3, 1:4 and 1:9, respectively. The gradient eluent was collected and concentrated. The same parts were combined after TLC monitoring. Five parts were obtained. Among them, the 3:2 part 68.4 g was further separated by column chromatography with methanol as the mobile phase. The part enriched with azacyclanones was further separated by column chromatography with 56% methanol as the mobile phase, the flow rate being 12 ml / min, the column being 2.12 x 250 mm, and the column temperature being 25°C. The eluent was collected and concentrated. The same parts were combined after TLC monitoring. The anti-tobacco mosaic virus active anthraquinone compound was obtained. µ The anti-tobacco mosaic virus active anthraquinone compound was obtained by column chromatography with Zorbax Prep HT GF reversed-phase preparative column as the stationary phase and UV detector with the detection wavelength being 408 nm, each time 0.8 mL of sample was injected, and the chromatographic peak was collected for 30.8 min. After multiple accumulations, the sample was evaporated to dryness to obtain the anti-tobacco mosaic virus active anthraquinone compound.

[0081] Example 4

[0082] Any of the compounds prepared in Examples 1-3 was a light red gum. The determination method was the same as that in Example 1. It was confirmed that the compound prepared in Examples 1-3 was the 5-methyl-3,6-dimethoxy-2-(3'-methyl-1'-pyrrol-2'-yl)anthracene-9,10-dione. H

[0083] Example 5

[0084] The anti-tobacco mosaic virus activity test was carried out by the half-leaf method. The anti-tobacco mosaic virus activity of the compound was determined at the mass concentration of the agent being 20 μM. On 5-6 tobacco plants, the suitable leaf for testing (normal leaf row, no disease and no insect) was selected. The leaf was evenly sprinkled with fine emery powder. The prepared tobacco mosaic virus source (3.0 x 10 -3 ​) The leaves were evenly spread on the sandpaper, and then were immediately put into the culture dish containing the drug solution for 20 min after all the selected leaves were treated with the virus. The leaves were taken out, and the water drops and the solution on the leaves were removed. Two half leaves were recovered and were placed in the enamel dish covered with glass, and the temperature was controlled at (23±2)℃. The dish was placed in the greenhouse under natural light. The dry spots could be observed after 2-3 days. Another half leaf of each treatment was used as the control. Another group of the commercial ningnanmycin was used as the comparison. The relative inhibition rate was calculated according to the following formula.

[0085] XI%=(CK-T) / CK x 100%

[0086] X: relative inhibition rate (%), CK: the number of dry spots of the half leaf soaked in water, T: the number of dry spots of the half leaf soaked in the solution.

[0087] The relative inhibition rate of the compound was 56.2%, which was higher than the relative inhibition rate of the control ningnanmycin (32.4%), indicating that the compound had good activity against tobacco mosaic virus.

Claims

1. A nitrogen-containing heterocyclic anthraquinone compound with activity against tobacco mosaic virus, characterized in that, The nitrogen-containing heterocyclic anthraquinone compounds with anti-tobacco mosaic virus activity were prepared from *Cassia tora* via pretreatment, extract extraction, MCI decolorization, silica gel column chromatography, and high-performance liquid chromatography. The molecular formula of the nitrogen-containing heterocyclic anthraquinone compounds with anti-tobacco mosaic virus activity is: C 22 H 19 NO4, named as: 5-methyl-3,6-dimethoxy-2-(3'-methyl-1'-) H 3,6-dimethoxy-5-methyl-2-(3'-methyl-1'-yl)anthracene-9,10-dione, also known as pyrrole-2'-yl)anthracene-9,10-dione. H -pyrrol-2'-yl)anthracene-9,10-dione has the following structure: 。 2. A method for preparing the nitrogen-containing heterocyclic anthraquinone compound with anti-tobacco mosaic virus activity as described in claim 1, comprising the following steps: pretreatment, extract extraction, MCI decolorization, silica gel column chromatography, and high-performance liquid chromatography separation, using the fine branches of the Dai medicinal herb *Cassia tora* as raw material. A. Pre-treatment: Crush the fine branches of Cassia biloba and pass them through a 20-60 mesh sieve to obtain material a; B. Extraction of extract: Add 2-6 times the mass of organic extraction solvent to material a, and extract by soaking at room temperature 2-5 times, each extraction time being 12-20 hours. Combine the extracts and filter. After concentrating the filtrate, add an equal volume of ethyl acetate for extraction 2-4 times, and concentrate to obtain sample extract b. The organic extraction solvent is a 70-100% (w / w) methanol aqueous solution, a 70-100% (w / w) ethanol aqueous solution, or a 65-100% (w / w) acetone aqueous solution. C. MCI decolorization: The sample extract b is concentrated and decolorized on an MCI column. The effluent is collected and concentrated under reduced pressure to obtain extract c. D. Silica gel column chromatography: 1) Add 3 to 10 times the mass of extract c to a column of 200 to 300 mesh silica gel and elute with a chloroform-methanol solution of volume ratio of 20:1 to 1:

1. Monitor by TLC and combine identical fractions. 2) The eluent obtained by elution with chloroform-methanol solution in an 8:2 ratio is packed into a column with 200-300 mesh silica gel in a volume equivalent to 2.5-10 times the mass of extract c. Further separation is carried out by silica gel column chromatography, followed by gradient elution with chloroform-acetone solution. The eluents are collected and monitored by TLC. The same fractions are combined. E. Gel column chromatography purification: The chloroform-acetone eluent in a 3:2 ratio was concentrated and then dissolved in methanol. Using methanol as the mobile phase, the mixture was purified again by Sephadex LH-20 dextran gel chromatography to obtain crude nitrogen-containing anthraquinone compounds with anti-tobacco mosaic virus activity. F. High-performance liquid chromatography separation: The crude product of the nitrogen-containing heterocyclic anthraquinone compound with anti-tobacco mosaic virus activity was separated and purified by high-performance liquid chromatography to obtain the nitrogen-containing heterocyclic anthraquinone compound with anti-tobacco mosaic virus activity. The high-performance liquid chromatography (HPLC) separation and purification was performed using a 21.2 mm × 250 mm, 5 μ m of ZorbaxPrepHT GF C 18 The chromatographic column was used with a flow rate of 12 mL / min, the mobile phase was 56 wt% methanol-water solution, the UV detector was set at a wavelength of 408 nm, and the injection depth was 0.5–2.0 μL per sample. m L, collect the eluent corresponding to the chromatographic peak retention time of 30.8 min after each injection and add it multiple times. After removing the solvent from the eluent, the nitrogen-containing anthraquinone compound of the present invention is obtained.

3. The preparation method according to claim 2, characterized in that, Before step D, silica gel column chromatography, the sample is diluted with 1.5 to 3 times the mass of extract c in acetone or methanol, and then mixed with 1.5 to 2.5 times the mass of extract c in 80 to 100 mesh silica gel.

4. The preparation method according to claim 2, characterized in that, In step D, the volume ratio of chloroform to methanol in the chloroform-methanol solution is 20:1, 9:1, 8:2, 7:3, 6:4, and 5:

5.

5. The preparation method according to claim 2, characterized in that, In step D, the volume ratio of chloroform to acetone in the chloroform-acetone solution is 4:1, 3:2, 2:3, 1:4, and 1:

9.

6. The application of the nitrogen-containing heterocyclic anthraquinone compound with anti-tobacco mosaic virus activity as described in claim 1, characterized in that, The application of the nitrogen-containing heterocyclic anthraquinone compounds with anti-tobacco mosaic virus activity in the preparation of anti-tobacco mosaic virus drugs.

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